Current Fungal Infection Reports https://doi.org/10.1007/s12281-018-0314-0

ADVANCES IN DIAGNOSIS OF INVASIVE FUNGAL INFECTIONS (S CHEN, SECTION EDITOR)

11. Liu, N.-N., Chi, Z., Liu, G.-L., Chen, T.-J., Jiang, H., Hu, J., and Chi, Z.-M. (2018). α-Amylase, glucoamylase and isopullulanase determine molecular weight of The Black : an Update on Species Identification and Diagnosis pullulan produced by Aureobasidium melanogenum P16. International Journal of 1 1 Biological Macromolecules 117: 727-734. Connie F. Cañete-Gibas & Nathan P. Wiederhold

12. Tang, R.-R., Chi, Z., Jiang, H., Liu, G.-L., Xue, S.-J., Hu, Z., and Chi, Z.-M. (2018). Overexpression of a pyruvate carboxylase gene enhances extracellular liamocin and # Springer Science+Business Media, LLC, part of Springer Nature 2018 intracellular lipid biosynthesis by Aureobasidium melanogenum M39. Process Abstract Biochemistry 69: 64-74. Purpose of review Black -like fungi are capable of causing a wide range of infections, including invasive disease. The diagnosis of infections caused by these species can be problematic. We review the changes in the nomenclature and of 13. Chen, T.-J., Chi, Z., Jiang, H., Liu, G.-L., Hu, Z., and Chi, Z.-M. (2018). Cell wall these fungi, and methods used for detection and species identification that aid in diagnosis. integrity is required for pullulan biosynthesis and glycogen accumulation in Recent findings Molecular assays, including DNA barcode analysis and rolling circle amplification, have improved our ability to Aureobasidium melanogenum P16. BBA - General Subjects 1862: 1516-1526. correctly identify these species. A proteomic approach using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has also shown promising results. While progress has been made with molecular techniques using direct specimens, data are currently limited.

Summary Molecular and proteomic assays have improved the identification of -like fungi. However, improved molecular and proteomic databases and better assays for the detection and identification in direct specimens are needed to improve the diagnosis of disease caused by black yeast-like fungi.

Keywords Black yeasts . Diagnosis . Species identification . . . Fonsecaea . Rhinocladiella

Introduction infections, which are associated with significant morbidity [7••, 8–12]. Significant differences in pathogenicity and viru- Black yeast-like fungi and their filamentous relatives are ca- lence have also been reported between closely related species, pable of causing a wide range of often difficult to treat infec- and this may be a function of the individual species’ ability to tions. Infections caused by members of the order grow at certain temperatures. Those species that are able to , under which these fungi fall, include myce- grow at human body temperature (i.e., 37 °C) or higher are toma, , and phaeohyphomycoses. capable of causing systemic or disseminated disease. These Hematogenously disseminated infections can also occur and species include but are not limited to E. dermatitidis, are primarily caused by Exophiala species that are able to E. jeanselmei, E. oligospora, Cladophialophora bantiana, produce yeast-like cells [1–3, 4•]. Although rare, case reports and Fonsecaea species [4•]. In addition to causing infections of endocarditis due to Exophiala species have been reported in in humans, black yeast-like fungi and their filamentous rela- the literature [5, 6], and a recent epidemiologic study also tives are also capable of causing disease in cold-blooded ani- reported one case caused by [7••]. mals [13]. Species that grow at temperatures between 35 and Other species are capable of causing central nervous system 37 °C often are associated with superficial and cutaneous in- fections in humans but may cause systemic disease in cold- blooded animals. In contrast, mesophilic species, which only grow at temperatures between 27 and 33 °C, are limited to This article is part of the Topical Collection on Advances in Diagnosis of cold-blooded animals, although infections in invertebrates Invasive Fungal Infections have also been reported [14]. * Connie F. Cañete-Gibas The diagnosis of invasive fungal infections remains chal- [email protected] lenging. In many patients with probable or presumed invasive mycoses, fungal cultures are rarely positive, thus hampering the diagnosis of these infections. However, it is known that 1 Testing Laboratory, Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at San Antonio, delays in diagnosis and initiation of appropriate therapy are 7703 Floyd Curl Drive, San Antonio, TX 78229, USA associated with worse outcomes in patients with invasive

Reproduced from Current Fungal Infection Reports (2018). https://doi.org: 10.1007/s12281-018-0314-0 Connie F. Cañete-Gibas: Participant of the 19th UM, 1991-1992.

220 221 Curr Fungal Infect Rep fungal infections [15–17]. Although these studies have pri- Table 1 List of medically important species in the Family marily involved patients with invasive and can- and Cyphellophoraceae [21–24] didiasis, it is presumed that delays in appropriate therapy Genus Species would also lead to worse outcomes in patients with infections caused by other fungi. This review discusses the species of Cladophialophora Cladophialophora arxii Cladophialophora bantiana black yeast-like fungi and their filamentous relatives, changes Cladophialophora boppii in their nomenclature, and methods for diagnosis and species Cladophialophora carrionii identification. (formerly Cladophialophora ajelloi) Cladophialophora devriesii Cladophialophora emmonsii Cladophialophora immunda Species of Black Yeast-like Fungi, Their Cladophialophora modesta Filamentous Relatives and Characteristics Cladophialophora mycetomatis Cladophialophora samoensis Cladophialophora saturnica Black yeast-like fungi and their filamentous relatives are Cladophialophora subtilits dematiaceous, non-lichenized fungi characterized by the pres- Cyphellophora Cyphellophora laciniata ence of a dark yeast-like phase that later becomes a mycelial Cyphellophora pluriseptata Cyphellophora suttonii stage [18]. These fungi exhibit slow to moderate growth and Exophiala Exophiala asiatica as previously noted are classified under the ascomycete order Exophiala attenuata Chaetothyriales (subphylum Pezizomycotina). Five families Exophiala bergeri Exophiala castellanii are recognized within this order: Chaetothyriaceae, Cyphellophoraceae, Epibryaceae, Herpotrichiellaceae, and (formerly Wangiella dermatitidis) Trichomeriaceae [19••, 20]. Most clinically important species Exophiala equina Exophiala halophila are included in the Family Herpotrichiellaceae and Cyphellophoraceae, mainly in the genera Cladophialophora, Exophiala lecanii-corni Cyphellophora, Exophiala, Fonsecaea, Phialophora, and Exophiala mesophila Exophiala oligosperma Rhinocladiella (Table 1)[19••]. Exophiala spinifera Cladophialophora species produce one-celled ellipsoidal Exophiala “siphonis” to fusiform dry conidia arising through blastic, acropetal Exophiala xenobiotica Fonsecaea Fonsecaea pedrosoi (formerly Fonsecaea conidiogenesis and arranged in branched chains [25]. The compacta, a dysgonic mutant or genus Cyphellophora is characterized by having melanized variety of Fonsecaea pedrosoi) thalli with phialidic condiogenesis, phialidic openings inserted Fonsecaea multimorphosa Fonsecaea nubica directly on hyphae or occasionally on flask-shaped Fonsecaea pedrosoi conidiogenous cells, and producing small clusters of oliva- Phialophora Phialophora Americana ceous, septate or non-septate, mostly curved conidia [26]. Phialophora europaea The genus Exophiala is comprised of species that exhibit Rhinocladiella Rhinocladiella aquaspera smooth or velvety, initially mucoid, black, olivaceous green (formerly Acrotheca aquaspersa) or dark brown colonies [27]. Nearly all species within this Rhinocladiella basitona genus are characterized and recognizable by their production (formerly Ramichloridium basitonum) Rhinocladiella mackenziei of budding cells, and the yeast-to-hyphae transition mostly (formerly Ramichloridium mackenziei) proceeds via torulose hyphae. These species are highly pleo- Rhinocladiella similis morphic, and morphologic characteristics are often deter- mined by factors such as culture media, incubation tempera- ture, and the age of the culture. The genus Fonsecaea includes smooth- and thin-walled, one or occasionally two-celled, el- species that are defined by the presence of indistinct mela- lipsoidal to clavate in shape [21, 30]. nized conidiophores with blunt, dispersed denticles that bear single or short chains of conidia that eventually become un- branched [28]. The genus Phialophora is characterized by Changes in Nomenclature melanized hyphae and by the production of slimy, one- celled conidia in heads from phialides with distinct collarettes Until 2013, polymorphic higher fungi (Dikarya) were allowed [29]. Another genus of black yeast-like fungi Rhinocladiella is to carry multiple names to describe their teleomorphic and comprised of species with sympodial conidiogenesis. The co- anamorphic stages. These stages occur independently, and nidiophores are dark brown, straight, upright, unbranched, their relationship was not always established. The wide use thick walled, and produce conidia that are sub-hyaline, of molecular methods has facilitated the establishment of

222 223 Curr Fungal Infect Rep Curr Fungal Infect Rep fungal infections [15–17]. Although these studies have pri- Table 1 List of medically important species in the Family relationships between these various stages in the life cycles of mutant or a morphological variety occurring in F. pedrosoi marily involved patients with invasive aspergillosis and can- Herpotrichiellaceae and Cyphellophoraceae [21–24] fungi, and has led to the abolition of the dual nomenclature [24, 36, 37]. didiasis, it is presumed that delays in appropriate therapy Genus Species system of fungi and the adoption of the One Fungus One The genus Ramichloridium was erected in 1937 by Stahel would also lead to worse outcomes in patients with infections Name system, as provided in the new “International Code of but was invalid due to the lack of a Latin diagnosis. This genus caused by other fungi. This review discusses the species of Cladophialophora Cladophialophora arxii Nomenclature for Algae, Fungi, and Plants” [31, 32]. The was resurrected by De Hoog in 1977 to accommodate species Cladophialophora bantiana black yeast-like fungi and their filamentous relatives, changes Cladophialophora boppii classification of fungi has now moved from phenotypic char- with erect, dark, more or less differentiated, branched or un- in their nomenclature, and methods for diagnosis and species Cladophialophora carrionii acteristics to phylogenetic relationships. Nucleic acid varia- branched conidiophores and predominantly aseptate conidia identification. (formerly Cladophialophora ajelloi) tion has proved a means of ensuring that genera are monophy- produced on a sympodially proliferating rachis [38], and in- Cladophialophora devriesii letic, and that species that differ at the ordinal and family level cluded Ramichloridium basitonum and Ramichloridium Cladophialophora emmonsii Cladophialophora immunda are no longer accepted as members of a single genus. The mackenziei. However, a re-evaluation of the genus Species of Black Yeast-like Fungi, Their Cladophialophora modesta advantage of the phylogenetic approach is that close relatives Ramichloridium using morphologic and DNA sequence data Filamentous Relatives and Characteristics Cladophialophora mycetomatis come together even if they are morphologically quite differ- resulted in the proper accommodation of these two species in Cladophialophora samoensis Cladophialophora saturnica ent. This approach may be useful for predictions of pathoge- the genus Rhinocladiella as Rhinocladiella mackenziei and Black yeast-like fungi and their filamentous relatives are Cladophialophora subtilits nicity or susceptibility to antifungals. Conversely, distant re- Rhinocladiella basitona [30]. dematiaceous, non-lichenized fungi characterized by the pres- Cyphellophora Cyphellophora laciniata lationships are expected to predict large differences in clini- ence of a dark yeast-like phase that later becomes a mycelial Cyphellophora pluriseptata cally relevant parameters [22]. Cyphellophora suttonii stage [18]. These fungi exhibit slow to moderate growth and Exophiala Exophiala asiatica Several black yeast-like fungi have been known by differ- Molecular Methods of Species Identification as previously noted are classified under the ascomycete order Exophiala attenuata ent names, and this has caused confusion within the medical Chaetothyriales (subphylum Pezizomycotina). Five families Exophiala bergeri mycology community. For example, carrionii Traditionally, phenotypic characteristics have been used as the Exophiala castellanii are recognized within this order: Chaetothyriaceae, Exophiala dermatitidis and Cladophialophora ajelloi were described separately. Due primary method of fungal species identification. For black Cyphellophoraceae, Epibryaceae, Herpotrichiellaceae, and (formerly Wangiella dermatitidis) to noticeable similarities in their basic morphology, Honbo yeast-like fungi, identification based on morphology alone Trichomeriaceae [19••, 20]. Most clinically important species Exophiala equina et al. conducted a study of their relationship and found that remains difficult. This is especially true for Exophiala species, Exophiala halophila are included in the Family Herpotrichiellaceae and Exophiala jeanselmei the Cladosporium and Phialophora synanamorphs produced which are pleomorphic and produce synanamorphs during Cyphellophoraceae, mainly in the genera Cladophialophora, Exophiala lecanii-corni by Cladosporium carrionii were identical to those produced their complex life cycles [39, 40]. The similarity of these Cyphellophora, Exophiala, Fonsecaea, Phialophora, and Exophiala mesophila by isolates of Cladophialophora ajelloi [23]. Cladosporium structures, even in distantly related species based on phyloge- Exophiala oligosperma Rhinocladiella (Table 1)[19••]. Exophiala spinifera carrionii was then emended to include the Phialophora netic analysis, and the lack of comparable physiological data, Cladophialophora species produce one-celled ellipsoidal Exophiala “siphonis” anamorph, and Cladophialophora ajelloi was considered to make it difficult to use morphologic criteria for reliable spe- to fusiform dry conidia arising through blastic, acropetal Exophiala xenobiotica be a later synonym. Cladosporium carrionii was formally cies identification [39]. Fonsecaea Fonsecaea pedrosoi (formerly Fonsecaea conidiogenesis and arranged in branched chains [25]. The compacta, a dysgonic mutant or redescribed and designated in an appropriate combination as DNA sequence analysis has proven to be a powerful tool genus Cyphellophora is characterized by having melanized variety of Fonsecaea pedrosoi) Cladophialophora carrionii [33]. for fungal species identification, including the identification of thalli with phialidic condiogenesis, phialidic openings inserted Fonsecaea multimorphosa Another example of changing nomenclature is that of opportunistic and pathogenic organisms. The internal tran- Fonsecaea nubica directly on hyphae or occasionally on flask-shaped Fonsecaea pedrosoi Exophiala dermatitidis. In 1937, Kano described the species scribed spacer (ITS) region of nuclear ribosomal RNA is a conidiogenous cells, and producing small clusters of oliva- Phialophora Phialophora Americana Hormiscium dermatitidis [34]. Due to its polymorphic nature, routinely used target for fungal species identification [41]. ceous, septate or non-septate, mostly curved conidia [26]. Phialophora europaea confusion was caused regarding its correct placement, and the ITS is commonly used for routine identification by doing sim- The genus Exophiala is comprised of species that exhibit Phialophora verrucosa species has undergone several taxonomic changes (i.e., ilarity searches in publicly available genetic databases, such as Rhinocladiella Rhinocladiella aquaspera smooth or velvety, initially mucoid, black, olivaceous green (formerly Acrotheca aquaspersa) Fonsecaea dermatitidis, Hormodendrum dermatitidis, GenBank, and phylogenetic analysis. BLAST similarity or dark brown colonies [27]. Nearly all species within this Rhinocladiella basitona Phialophora dermatitidis). The genus Wangiella was erected to search results should be considered carefully, as sequences genus are characterized and recognizable by their production (formerly Ramichloridium basitonum) accommodate Hormiscium dermatitidis, as it was found to be in publicly available databases may be assigned the wrong Rhinocladiella mackenziei of budding cells, and the yeast-to-hyphae transition mostly (formerly Ramichloridium mackenziei) invalidly published because of the lack of a Latin diagnosis. taxon due to sample contamination, chimerism, poor taxo- proceeds via torulose hyphae. These species are highly pleo- Rhinocladiella similis Thus, the species was renamed as Wangiella dermatitidis [34]. nomic identification, and other errors [42]. Often, the morphic, and morphologic characteristics are often deter- Morphologically, Wangiella dermatitidis is intermediate between mislabeled sequences may be corrected in the published man- mined by factors such as culture media, incubation tempera- PhialophoraandExophiala[34],anditwasformallytransferredto uscript but the correction may not occur within the database. ture, and the age of the culture. The genus Fonsecaea includes smooth- and thin-walled, one or occasionally two-celled, el- the genus Exophiala as Exophiala dermatitidis based on the ab- In black yeast-like fungi, the ITS region exhibits low var- species that are defined by the presence of indistinct mela- lipsoidal to clavate in shape [21, 30]. sence of phialides with collarettes [27]. iability among species and is not useful for discriminating nized conidiophores with blunt, dispersed denticles that bear was first described as between them [18, 43–45]. Other DNA targets, such as the single or short chains of conidia that eventually become un- Hormodendrum compactum by Carrion in 1935 from a clini- nuclear ribosomal gene (small and large subunits), elongation branched [28]. The genus Phialophora is characterized by Changes in Nomenclature cal case [35], who observed that although it resembled factor α-1, and actin have been used in conjunction with ITS melanized hyphae and by the production of slimy, one- Hormodendrum pedrosoi (now known as Fonsecaea to improve sensitivity [13, 19••, 39, 45]. Heinrich et al. ob- celled conidia in heads from phialides with distinct collarettes Until 2013, polymorphic higher fungi (Dikarya) were allowed pedrosoi), H. compactum was morphologically distinct, and served that the ITS2 region of species in the Family [29]. Another genus of black yeast-like fungi Rhinocladiella is to carry multiple names to describe their teleomorphic and was given the species epithet compactum due to the compact Herpotrichiellacaea contains homopolymeric regions mostly comprised of species with sympodial conidiogenesis. The co- anamorphic stages. These stages occur independently, and arrangement of the conidia. Subsequent studies found that the of poly (T) that may account for approximately 8% of artificial nidiophores are dark brown, straight, upright, unbranched, their relationship was not always established. The wide use morphologic separation of F.compacta does not coincide with variances among species [39]. To avoid this problem, barcode thick walled, and produce conidia that are sub-hyaline, of molecular methods has facilitated the establishment of molecular data, and that this is no more than a dysgonic identifiers within domain 2 of ITS2 were developed. Domain

222 223 Curr Fungal Infect Rep

2 of ITS2 provides highly conserved flanking sequences with proteins. The spectra that are obtained represent a unique finger- a highly variable section in between, which varies markedly in print, known as a peptide mass fingerprint, and are compared to a both nucleotide composition and length between closely relat- library of spectra of known species in order to make the identi- ed taxonomic entities. These barcode identifiers have been fication [55, 56]. MALDI-TOF MS has been found to be a rapid found to be reliable, easy to use, and highly practicable for and sensitive method for species identification of bacteria, yeast, the rapid identification of clinically important black yeast-like and filamentous fungi [54•, 57–61] and may be advantageous to fungi and closely related species [39]. DNA sequence analysis due to rapid turn-around-time and the Species identification of black yeast-like fungi has been relatively low cost of consumables. Independent studies have improved with the introduction of barcode identifiers using demonstrated that this method is capable of identifying and dis- the ITS2 region. Although barcode identifiers are useful, this criminating between different Exophiala species, as well as other method still involves sequencing of the ITS2 region, which is related species [54•, 61, 62]. In a recent multicenter study, 31 of relatively expensive and does not have a rapid turn-around- 31 E. dermatitidis isolates were correctly identified to the species time. Rolling circle amplification is an isothermal amplifica- level [54•]. Good results were also observed with tion method that uses padlock probes that hybridize to target Cladophialophora bantiana where 28 of 29 isolates were cor- DNA or RNA [46]. Two ends of the probe become juxtaposed rectly identified. Interestingly, one C. bantiana isolate was and can be joined by a DNA ligase when both ends of the misidentified as a Candida collicculosa. Of note, 7 of the 32 probes show perfect complementarity. This allows for the de- E. xenobiotica isolates evaluated were not identified by this par- tection of single nucleotide mismatches and prevents non- ticular assay despite confirmation of all species identifications by specific amplification [46, 47]. Najafzadeh et al. reported that DNA sequence analysis. Similar success in the identification of this technique could discriminate between Exophiala species E. dermatitidis and other Exophiala species has been reported by with correct identification achieved unambiguously [46]. others with the use of MALDI-TOF MS technology [61, 63]. In Given that this method is more rapid than DNA sequence one study that included 89 isolates, 83 corresponded to 16 known analysis, it has potential to be used as an assay in clinical species of Exophiala and Rhinocladiella, and the remaining 6 laboratories for fungal species identification. represented two novel Exophiala species based on phylogenetic Other molecular approaches using PCR-based assays have analysis [63]. A larger study that included 110 Exophiala isolates been used in the identification of black yeast-like fungi. One reported identification rates to the genus or species level between approach utilized a novel species-specific primer pair (i.e., 80.6 and 100% for most species [62]. Thus, MALDI-TOF MS Exdf, Exdr) that targeted the conserved regions of the partial appears to be a robust assay for the identification of Exophiala ITS1-complete 5.8S-partial ITS2 rRNA region of species, although more work is needed for other species. E. dermatitidis [48]. When tested on presumptive black yeast isolates cultured from sputum samples in a cohort of cystic fibro- sis patients, the primers were found to be specific, as cultures Detection and Identification in Direct were confirmed to be E. dermatitidis by DNA sequence analysis. Specimens Another method specific for the detection of E. jeanselmei, termed Ejeanselmei_ITS qPCR, which utilizes SYBR green A major limitation of DNA sequence analysis and MALDI- chemistry and real-time PCR, was shown to be 100% sensitive TOF MS is the need for isolates growing in culture, as many and specific for this particular species [49]. Although tested on patients with invasive fungal infections, including those water samples, this method has the potential to be applied rou- caused by black yeast-like fungi, may be culture negative. tinely for the detection and identification of Exophiala species This has clinical implications as clinicians may be forced to and other black yeast-like fungi in clinical specimens. use empiric therapy for a presumptive fungal infection, and the drugs that are used may be suboptimal against the actual infecting organism. Surrogate marker assays are available and Matrix-Assisted Laser Desorption/Ionization are often used to aid the diagnosis of certain invasive fungal Time-of-Flight Mass Spectrometry infections. These include the 1,3-β-D-glucan assay and the Aspergillus galactomannan assay, which are capable of being Another method that has demonstrated great potential for the performed on biological fluids, such as serum and bronchoal- species identification of various fungi, including black yeast- veolar lavage fluid. The 1,3-β-D-glucan assay detects the like species, is that of matrix-assisted laser desorption/ presence of 1,3-β-D-glucan polymers, a major component of ionization time-of-flight mass spectrometry (MALDI-TOF the cell wall of numerous pathogenic fungi, including black MS), which has proven to be a timely, accurate, and reproducible yeast-like organisms [64]. However, this pan-fungal assay is method for the identification of different microbes [50–53, 54•]. incapable of determining which fungal species may be caus- Species identification using this method is based on the detection ing the infection, and false-positive reactions have been re- of mass-to-charge (m/z) ratios of protonated ions from ribosomal ported with its use [65]. In contrast, the galactomannan assay

224 225 Curr Fungal Infect Rep Curr Fungal Infect Rep

2 of ITS2 provides highly conserved flanking sequences with proteins. The spectra that are obtained represent a unique finger- is specific for Aspergillus species and is unable to detect black as cold-blooded animals. Marked changes in the nomenclature a highly variable section in between, which varies markedly in print, known as a peptide mass fingerprint, and are compared to a yeast-like fungi and their filamentous relatives. and taxonomy of these species has also occurred. The diagno- both nucleotide composition and length between closely relat- library of spectra of known species in order to make the identi- Another approach for the detection and identification of fungi sis of infections caused by these species can be problematic, ed taxonomic entities. These barcode identifiers have been fication [55, 56]. MALDI-TOF MS has been found to be a rapid in direct specimens is the use of PCR-based assays on tissue, and species identification based solely on morphologic fea- found to be reliable, easy to use, and highly practicable for and sensitive method for species identification of bacteria, yeast, including fresh tissue and paraffin-embedded specimens. This tures may be fraught with difficulty due to similarities in char- the rapid identification of clinically important black yeast-like and filamentous fungi [54•, 57–61] and may be advantageous to approach may be especially useful where cultures are negative, acteristics among different species. Discrimination between fungi and closely related species [39]. DNA sequence analysis due to rapid turn-around-time and the but histopathology is consistent with a fungal infection. These closely related species can be accomplished by DNA sequenc- Species identification of black yeast-like fungi has been relatively low cost of consumables. Independent studies have assays have primarily targeted one or more regions within the ing, including DNA barcode analysis based on the ITS2 rRNA improved with the introduction of barcode identifiers using demonstrated that this method is capable of identifying and dis- rRNA cluster, including the 18S, 28S, and ITS regions, as these region. MALDI-TOF MS has also proven to be useful in the the ITS2 region. Although barcode identifiers are useful, this criminating between different Exophiala species, as well as other areas contain both highly conserved and variable regions, and are species identification of Exophiala species, although more method still involves sequencing of the ITS2 region, which is related species [54•, 61, 62]. In a recent multicenter study, 31 of present in multiple copies, thus improving the sensitivity of these work is needed to determine its utility for other species. relatively expensive and does not have a rapid turn-around- 31 E. dermatitidis isolates were correctly identified to the species assays [66••]. Studies have reported some success with this ap- Work was also done to assess the utility of molecular ap- time. Rolling circle amplification is an isothermal amplifica- level [54•]. Good results were also observed with proach in the identification of black yeast-like fungi, although the proaches for the detection of fungal species within direct spec- tion method that uses padlock probes that hybridize to target Cladophialophora bantiana where 28 of 29 isolates were cor- data are quite limited. In one study, Exophiala species were de- imens. However, available data for Exophiala species and DNA or RNA [46]. Two ends of the probe become juxtaposed rectly identified. Interestingly, one C. bantiana isolate was tected in three culture-positive specimens, including synovial other related fungi are limited. and can be joined by a DNA ligase when both ends of the misidentified as a Candida collicculosa. Of note, 7 of the 32 fluid from one patient and skin from two others, with the use probes show perfect complementarity. This allows for the de- E. xenobiotica isolates evaluated were not identified by this par- of a pan-fungal PCR assay that targeted the ITS1 region [43, Compliance with Ethical Standards tection of single nucleotide mismatches and prevents non- ticular assay despite confirmation of all species identifications by 66••]. Interestingly, one E. jeanselmei identified by morphologic specific amplification [46, 47]. Najafzadeh et al. reported that DNA sequence analysis. Similar success in the identification of characteristics in a skin specimen was found to be a E. spinifera Conflict of Interest Connie F. Cañete-Gibas declares no conflict of this technique could discriminate between Exophiala species E. dermatitidis and other Exophiala species has been reported by based on the results of the molecular assay, while the other interest. Nathan P. Wiederhold has received grants from bioMerieux, Astellas, with correct identification achieved unambiguously [46]. others with the use of MALDI-TOF MS technology [61, 63]. In E. jeanselmei identified by morphology was reported only to Pfizer, Merck, Viamet, F2G, MOE Medical Devices, and Cidara and has Given that this method is more rapid than DNA sequence one study that included 89 isolates, 83 corresponded to 16 known the genus level (Exophiala) based on the DNA sequence results. received travel reimbursement from Gilead. analysis, it has potential to be used as an assay in clinical species of Exophiala and Rhinocladiella, and the remaining 6 A recent study using a similar approach but targeting the ITS2 laboratories for fungal species identification. represented two novel Exophiala species based on phylogenetic and D2 region of 28S rRNA in sterile specimens where histopa- Human and Animal Rights and Informed Consent This article does not Other molecular approaches using PCR-based assays have analysis [63]. A larger study that included 110 Exophiala isolates thology was consistent with fungal infection also reported good contain any studies with human or animal subjects performed by any of the authors. been used in the identification of black yeast-like fungi. One reported identification rates to the genus or species level between success [67]. However, only one patient with an infection due to approach utilized a novel species-specific primer pair (i.e., 80.6 and 100% for most species [62]. Thus, MALDI-TOF MS Exophiala species was included. Exdf, Exdr) that targeted the conserved regions of the partial appears to be a robust assay for the identification of Exophiala Several molecular-based platforms are now clinically avail- ITS1-complete 5.8S-partial ITS2 rRNA region of species, although more work is needed for other species. able for the detection and identification of yeasts within the References E. dermatitidis [48]. When tested on presumptive black yeast bloodstream. These include the Yeast Traffic Light and isolates cultured from sputum samples in a cohort of cystic fibro- QuickFISH assays (AdvanDx) [68, 69], the FilmArray blood Papers of particular interest, published recently, have been sis patients, the primers were found to be specific, as cultures Detection and Identification in Direct culture identification assay (bioMerieux) [70], and the T2 highlighted as: were confirmed to be E. dermatitidis by DNA sequence analysis. Specimens Candida magnetic resonance assay (T2Biosystems) [71, 72]. • Of importance Another method specific for the detection of E. jeanselmei, However, none of these assays is capable of detecting and •• Of major importance termed Ejeanselmei_ITS qPCR, which utilizes SYBR green A major limitation of DNA sequence analysis and MALDI- identifying Exophiala species, even though these fungi have chemistry and real-time PCR, was shown to be 100% sensitive TOF MS is the need for isolates growing in culture, as many yeast-like structures and can disseminate via the hematoge- 1. Nachman S, Alpan O, Malowitz R, Spitzer ED. Catheter-associated due to Wangiella (Exophiala) dermatitidis. J Clin and specific for this particular species [49]. Although tested on patients with invasive fungal infections, including those nous route. One assay, the PLEX-ID system, showed promise Microbiol. 1996;34(4):1011–3. water samples, this method has the potential to be applied rou- caused by black yeast-like fungi, may be culture negative. in the detection and identification of a large number of differ- 2. Al-Obaid I, Ahmad S, Khan ZU, Dinesh B, Hejab HM. Catheter- tinely for the detection and identification of Exophiala species This has clinical implications as clinicians may be forced to ent fungi, including Exophiala and Fonsecaea species by associated fungemia due to Exophiala oligosperma in a leukemic and other black yeast-like fungi in clinical specimens. use empiric therapy for a presumptive fungal infection, and combining multiplex PCR with electrospray ionization-mass child and review of fungemia cases caused by Exophiala species. the drugs that are used may be suboptimal against the actual spectrometry in order to amplify and identify organisms based Eur J Clin Microbiol Infect Dis. 2006;25(11):729–32. 3. Nucci M, Akiti T, Barreiros G, Silveira F, Revankar SG, Wickes infecting organism. Surrogate marker assays are available and on the mass-to-charge ratio of the PCR amplicons [73–75]. BL, et al. Nosocomial outbreak of Exophiala jeanselmei fungemia Matrix-Assisted Laser Desorption/Ionization are often used to aid the diagnosis of certain invasive fungal This assay was under clinical development for the detection of associated with contamination of hospital water. Clin Infect Dis. Time-of-Flight Mass Spectrometry infections. These include the 1,3-β-D-glucan assay and the up to 75 different fungal species and had shown promising 2002;34(11):1475–80. Aspergillus galactomannan assay, which are capable of being results with direct specimens [75]. Unfortunately, its develop- 4.• Seyedmousavi S, Netea MG, Mouton JW, Melchers WJ, Verweij Another method that has demonstrated great potential for the performed on biological fluids, such as serum and bronchoal- ment system has been discontinued by the manufacturer [76]. PE, de Hoog GS. Black yeasts and their filamentous relatives: prin- ciples of pathogenesis and host defense. Clin Microbiol Rev. species identification of various fungi, including black yeast- veolar lavage fluid. The 1,3-β-D-glucan assay detects the 2014;27(3):527–42. Comprehensive review of the black yeasts like species, is that of matrix-assisted laser desorption/ presence of 1,3-β-D-glucan polymers, a major component of and their filamentous relatives. ionization time-of-flight mass spectrometry (MALDI-TOF the cell wall of numerous pathogenic fungi, including black Conclusions 5. Ventin M, Ramirez C, Garau J. Exophiala dermatitidis de Hoog MS), which has proven to be a timely, accurate, and reproducible yeast-like organisms [64]. However, this pan-fungal assay is from a valvular aortal prothesis. Mycopathologia. 1987;99(1):45–6. method for the identification of different microbes [50–53, 54•]. incapable of determining which fungal species may be caus- Black yeast-like fungi and their filamentous relatives are ca- 6. Patel AK, Patel KK, Darji P, Singh R, Shivaprakash MR, Chakrabarti A. Exophiala dermatitidis endocarditis on native aortic Species identification using this method is based on the detection ing the infection, and false-positive reactions have been re- pable of causing a wide range of mycoses, and can cause valve in a postrenal transplant patient and review of literature on of mass-to-charge (m/z) ratios of protonated ions from ribosomal ported with its use [65]. In contrast, the galactomannan assay disease in humans and other warm-blooded animals, as well E. dermatitidis infections. Mycoses. 2013;56(3):365–72.

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